The present application claims priority under 35 U.S.C. xc2xa7119(a) to German Priority Document No. 197 11 933.6, filed on Mar. 21, 1997.
The field of the invention is compounds which affect proliferation and differentiation of bone cells.
Bone differentiation is a complex process that involves the proliferation of osteoblasts and the formation of bone matrix. During childhood, a pronounced remodeling of bone takes place to allow skeletal growth, and remodeling continues throughout life. A controlled balance of bone destruction and new bone formation is essential for the maintenance of a specific bone mass. A reduced ability to produce new bone results in an overall loss of bone mass, osteoporosis, which represents a major medical problem, in particular, for post-menopausal women.
Osteogenesis in vivo and in various tissue culture systems may be separated into three consecutive stages. The early proliferation stage is characterized by DNA synthesis and the expression of genes, the gene products of which play a role in the formation of extracellular matrix, including collagen type 1, fibronectin and TGF-xcex2. The second stage of matrix maturation is characterized by a high expression of alkaline phosphatase which may condition the osteoid matrix for the third stage, characterized by synthesis of osteocalcin.
Processes of bone differentiation are mediated by a large number of growth factors and cytokines, many of which accumulate in calcified bone matrix. A key event controlling the extent of new bone formation is the transition of proliferating osteoblasts to resting bone cells which deposit bone matrix. The signals inducing this maturation process have not been identified up to now and are vaguely designated as factors involved in bone matrix maturation. Knowledge of such factors would make it possible to reduce or even entirely inhibit osteopetrotic growth or, inversely, stimulate proliferation of preosteoblasts, e.g., to attenuate osteoporotic symptoms.
T1-M is a receptor closely related to interleukin-1 (IL-1) receptor, but lacking affinity for IL-1 ligand. Up to now, the ligand binding to T1-M has not exactly been known, but two possible ligands have recently been described (Kumar, 1995; Gayle, 1996). The biological functions of these ligands remain to be elucidated. The receptor T1-M is always membrane-associated and is constitutively present on hematopoietic cells of the erythroid and mast cell lineages. It represents 2-5% of the total T1 protein produced (T1:T1-M and T1-S). By contrast, an extracellular soluble variant, the ligand binding domain of the complete T1-M receptor, T1-S, is secreted to the environment.
However, T1-S, is not a cleavage product of T1-M, but is expressed by differential splicing of T1 RNA in differentiating tissues, including bones and mammary glands. T1-S represents about 95% of the produced T1 protein; it is deposited in newly formed bone matrix. The T1-S and T1-M proteins as well as the gene coding for the two proteins have been described in detail in Klemenz (1989), Yanagisawa (1993) and Thomassen (1995) for mouse and in Tominaga (1992) for the human protein. Each of these references is hereby incorporated by reference herein.
There is a long felt need for the identification and development of agents which affect the proliferation and differentiation of bone cells. The present invention satisfies this need.
The invention relates to a composition comprising a compound capable of directly or indirectly modulating the activity of endogenous T1-M protein, the compound optionally containing one or more physiologically compatible additives.
In one aspect, the compound influences the activity of the T1-M protein by modulating the expression thereof.
In another aspect, the compound influences the activity of the T1-M protein by interacting with the T1-M protein.
In yet another aspect, the compound is capable of reducing the activity of the T1-M protein.
In a further aspect, the compound is a T1-M antisense nucleic acid. IN a preferred embodiment, the antisense nucleic acid is complementary to a portion of T1-M mRNA. In another preferred embodiment, the antisense nucleic acid is complementary to the 5xe2x80x2 region of T1-M ORF. In yet another preferred embodiment, the antisense nucleic acid is a DNA oligonucleotide having a sequence complementary to a DNA sequence selected from nucleotides 191-1201 and 277-796 of the cDNA sequence of T1-M ORF.
In another aspect, the compound is a T1-S-sense nucleic acid. In a preferred embodiment, compound is a T1-S mRNA.
In yet another aspect, the compound is an inhibitor which inhibits the function of the T1-M protein. In a preferred embodiment, the inhibitor is selected from the group consisting of a monoclonal antibody, a polyclonal antibody and a fragment of an antibody.
In a further aspect, the compound influences the activity of the T1-M protein by binding a T1-M protein ligand.
In another aspect, the compound is an expression vector which is capable of expressing T1-S. In a preferred embodiment, the compound is a competitive inhibitor of T1-M protein ligand binding. In another preferred embodiment, the competitive inhibitor is T1-S protein.
In yet another aspect, the compound enhances the activity of the T1-M protein. In a preferred embodiment, the compound is an expression vector capable of expressing T1-M. In yet another preferred embodiment, the compound stimulates the differentiation of bone cells. Also preferably, the compound is a T1-M protein ligand. More preferably, the ligand is an 18 kd protein expressed in BALB/c-3T3 cells.
In another aspect of the invention, the compound is in solution form.
In yet another aspect, the composition is prepared as a depot.
Also included in the invention is a method of influencing the proliferation and/or differentiation of bone cells. The method comprises administering to the cells a compound capable of modulating the activity of T1-M protein.
In one aspect of this method, modulation of the activity of the T1-M protein is effected by modulating the expression of the T1-M protein. In a preferred embodiment, modulation of the activity of the T1-M protein is effected by the interaction of T1-M protein with the compound. In another preferred embodiment, the activity of T1-M protein is reduced.
In one aspect, the compound is a T1-S-antisense nucleic acid and in another aspect, the compound is a T1-S-sense nucleic acid.
In another aspect, the compound is a T1-M protein inhibitor. In a preferred embodiment, the inhibitor is selected from the group consisting of a monoclonal antibody, a polyclonal antibody and a fragment of an antibody. In another preferred embodiment, the inhibitor is a competitive inhibitor. Preferably, the competitive inhibitor is T1-S protein.
In yet another aspect, the activity of T1-M protein is increased. Preferably, the compound effects a stimulation in the differentiation of the bone cells. Also preferably, the compound is a T1-M ligand. In a preferred embodiment, the ligand is an 18 kD protein expressed in BALB/c-3T3 cells.
The invention also relates to a method of modulating the activity of T1-M protein in vitro. The method comprises administering to a population of bone cells in culture a compound capable of modulating the activity of the T1-M protein. In one preferred embodiment, the activity of the T1-M protein is reduced, whereby proliferation of the bone cells is stimulated. In another preferred embodiment, the activity of the T1-M protein is increased, whereby differentiation of the bone cells is stimulated.
Also included in the invention is a method of treating osteoporosis in a human patient, the method comprising administering to the patient a compound capable of modulating the activity of T1-M protein.
In addition, the invention includes a method of treating osteopetrotic disease in a human patient, the method comprising administering to the patient a compound capable of modulating the activity of T1-M protein.